DESCRIPTION: (Applicant's Description) This project will investigate cellular responses to photodynamic therapy (PDT) sensitized by the phthalocyanine photosensitizer Pc 4 to test the hypothesis that mitochondrial membranes are sensitive targets of PDT directly activating apoptosis, whereas some extra-mitochondrial stress pathways modulate the mitochondrial events and apoptosis. In Aim 1, phthalocyanines synthesized by Project 1 that are of varying effectiveness will be examined by laser scanning confocal microscopy for their sub-cellular localization in mitochondria. Re-localization of Pc 4 during irradiation and binding of Pc 4 and analogs to the mitochondrial benzodiazepine receptor will also be evaluated. For Aim 2, the mechanism for activation of the mitochondrial pathway of apoptosis by Pc 4-PDT will be examined, including the formation of reactive oxygen, loss of mitochondrial membrane potential (delta psi mu), inhibition of respiration, and release of cytochrome c into the cytosol. Aim 3 will assess the role of cytochrome c-initiated steps of apoptosis after Pc 4-PDT by measuring the activation of caspase-3 and cleavage of some of its substrates, e.g., DNA Fragmentation Factor-45 and poly(ADP-ribose) polymerase (PARP). Tumor cells lacking caspase-3 and extracts from them will be supplemented with cloned hamster procaspase-3 and examined for reversal of defective apoptosis. In Aim 4, the role of stress kinases in PDT-induced cell killing will be assessed by manipulating the activity of two pathways that promote apoptosis. For Aim 5, PDT-induced apoptosis will be studied in tumors. In human A431 xenografts, we will investigate whether apoptotic cells of tumors are of human origin by staining with a human chromosome- specific probe or antibodies specific for human protein. Tumors derived from RIF-1 cells transfected with a hamster procaspase-3 gene will be compared to tumors from parental RIF-1 cells with respect to the incidence of apoptosis and overall tumor response. The proposed investigation will contribute to the understanding of cellular responses to Pc 4-PDT and their role in the fate of PDT-treated tumors. The results should suggest new opportunities for monitoring and modifying PDT responses, especially apoptosis, in normal and tumor tissue.